'Loop' domain deletional mutant of Bcl-x(L) is as effective as p29Bcl-x(L)in inhibiting radiation-induced cytosolic accumulation of cytochrome C (cyt c), caspase-3 activity, and apoptosis

Purpose/Objective: To investigate the effect of the enforced expression of p29Bcl-x(L) or its loop deletional mutant, p18Bcl-x(L)Delta, on irradiation -induced apoptosis and cell-cycle distribution of HL-60 cells. Materials & Methods: We compared the irradiation-induced molecular cascade of apoptosis in control human AML HL-60/neo versus Bcl-x(L) overexpressing (similar to 8-fold) (HL-60/Bcl-x(L)) and HL-60/Bcl-x(L)Delta cells that exp ress the loop domain deletional mutant construct (Delta 26-83 AA) of Bcl-x( L). The three cell lines were irradiated with 6MV photons to varying doses up to 20 Gy. Following this, cytosolic cyt c levels, caspase-3 activity, an d the Bcl-2 family of proteins were evaluated utilizing Western blot analys is (whole cell lysate or cytosolic S-100 fraction). Apoptosis was assessed by internucleosomal DNA fragmentation, Annexin-V staining and FACS analysis , as well as by morphologic criteria. The cell-cycle effects of radiation w ere analyzed by flow cytometry. Results: Eight hours following irradiation (12 Gy) of HL-60/neo cells, a ma rked increase (similar to 8-fold) in the cytosolic accumulation of cyt c in the S-100 fraction was observed, This was associated with the cleavage of caspase-3, as well as the generation of its poly (ADP-ribose) polymerase (P ARP) and DFF (DNA fragmentation factor)-45 cleavage activity. Twenty-four t o forty-eight hours after irradiation, internucleosomal DNA fragmentation a nd positive Annexin-V staining (32.3 +/- 3.3%) was detected in HL-60/neo ce lls. In contrast, in both HL60/Bcl-x(L) and HL80/Bcl-x(L)Delta cells, a sig nificantly lower percentage of apoptotic cells (p < 0.05) were detected and internucleosomal DNA fragmentation was not induced. Following irradiation, Western analysis neither demonstrated any significant alteration in Bcl-2, p29Bcl-x(L), p18Bcl-x(L)Delta, or Bax; nor induced CD95 (Fas receptor) or Fas ligand expression in any cell type. However, in all cell types, irradia tion produced approximately a 2-fold increase in the percentage of cells in the G(2)/M phase of the cell cycle. Conclusion: These results demonstrate that an intact loop domain is not nec essary for the full antiapoptotic function of Bcl-x(L) against irradiation- induced cytosolic accumulation of cyt c, caspase activation, and apoptosis of HL-60 cells. Additionally, the cell-cycle effects of ionizing radiation in HL-60 cells are not affected by enforced expression of Bcl-x(L) or Bcl-x (L)Delta. (C) 1999 Elsevier Science Inc.